Process for isolating and purifying ambrox

ABSTRACT

A method of isolating and purifying (−)-Ambrox from a reaction mixture comprising (−)-Ambrox and one or more of the compounds (II), (III) and (IV)

The present invention is concerned with a method of preparing, isolatingand purifying the isomer (−)-Ambrox. More particularly, the invention isconcerned with a method of preparing (−)-Ambrox by means of abioconversion, as well as to a method of its recovery and purificationfrom a reaction mixture.

AMBROFIX™ is the proprietary Givaudan trade name of the enantiomericallypure compound (−)-Ambrox, which has the general formula (I).

AMBROFIX™ is a very important molecule in the perfumers' palette ofingredients. It delivers a highly powerful, highly substantive andhighly stable ambery note for use in all perfumery applications.AMBROFIX™, available from Givaudan, is the most suitable material forobtaining an authentic ambergris odour note.

Currently, AMBROFIX™ is produced synthetically from starting materialsof natural origin. The availability and quality of certain startingmaterials are dependent on climatic conditions, as well associo-economic factors. Furthermore, since starting materials may beextracted from natural resources, with modest yields, they are availableat prices that will, in all likelihood, increasingly render their useuneconomical on an industrial scale. Accordingly, if commercialindustrial supplies of AMBROFIX™ are to continue to be available at areasonable cost, there is a need for a more cost-effective process ofproduction and purification, which is capable of industrialization.

An industrially scalable biotechnological route into AMBROFIX™ would beattractive because it is potentially less complex and less pollutingthan fully synthetic procedures.

A potentially useful substrate on which to attempt a bioconversion toprovide (−)-Ambrox is homofarnesol. In their seminal paper, Neumann etal (Biol. Chem. Hoppe-Seyler Vol. 367 pp 723-726 (1986)) discussed thefeasibility of converting homofarnesol to (−)-Ambrox under enzymaticcatalysis, employing the enzyme Squalene Hopene Cyclase (SHC). Thehomofarnesol employed was a mixture of the four geometric isomers ofthis molecule. Of the four isomers, only the 7E,3E geometric isomer(using conventional nomenclature) could be cyclized, and then only withvery low yield of the desired (−)-Ambrox.

JP 2009-60799 (Kao) discloses a synthesis whereby SHC acts on ahomofarnesol substrate to produce (−)-Ambrox. The substrate is a mixtureof all four geometric isomers (3Z,7Z; 3E,7Z; 3Z,7E; and 3E,7E). Thedocument only discloses the preparation of (−)-Ambrox from homofarnesolextracts containing SHC. The homofarnesol mixture is converted to(−)-Ambrox and its 9-epi stereoisomer, and purification can be carriedout by distillation or by column chromatography. Kao does not describe aprocess whereby homofarnesol is converted into (−)-Ambrox using intactmicroorganisms producing SHC, and furthermore, it does not provide anytechnical teaching related to the downstream processing of complexreaction mixtures obtained by such processes that can yield (−)-Ambroxin olfactively pure form.

To the applicant's knowledge, the prior art does not describe anyviable, industrially scalable processes, involving the SHC-catalyzedbioconversion of homofarnesol, to provide (−)-Ambrox in olfactively pureform.

Furthermore, if bioconversion of homofarnesol is to be realized on anindustrial scale, cost-efficient sources of highly pure,3E,7E-homfarnesol should be available. However, although syntheticroutes into homofarnesol have been described in the literature (see forexample US 2013/0273619), to the applicant's knowledge there are nocost-effective, industrial-scale sources of pure 7E,3E-homofarnesolcurrently available.

There remains a need to provide an economically feasible andindustrially scalable route into the valuable fragrance ingredient(−)-Ambrox.

In co-pending patent applications PCT/EP2014/072891 (published asWO2015/059293) and PCT2014/EP/072882 (published as WO2015/059290), theapplicant describes an efficient method of preparing7E,3E/Z-homofarnesol mixture that is enriched in the 7E,3E geometricisomer. The 7E,3E/Z-homofarnesol mixture is prepared frombeta-farnesene, and the isomeric information contained in this startingmaterial is preserved, such that the homofarnesol double bond at the7-position is fixed in the E-configuration. However, even 30 thiselegant chemistry still results in a 3E/Z isomer mixture. Pure7E,3E-homofarnesol remains synthetically challenging, and might only beachieved by means of economically disadvantageous purification ofisomeric mixtures.

Surprisingly, the applicant has found that 7E,3E/Z-homofarnesol mixturescan undergo a bioconversion process, whereby the homofarnesol mixture isenzymatically cyclized in the presence of a recombinant microorganismexpressing an enzyme, in particular a Squalene Hopene Cyclase (SHC)biocatalyst capable of bioconverting homofarnesol to (−)-Ambrox, toyield a reaction mixture from which (−)-Ambrox can be isolated inolfactively pure form with surprisingly facile downstream processing.

In one aspect of the invention there is provided the enzyme-catalyzedcyclisation of homofarnesol to provide a reaction mixture comprising(−)-Ambrox, wherein the homofarnesol comprises a mixture of7E,3E/Z-geometric isomers of homofarnesol, and wherein the reaction iscarried out in the presence of a recombinant microorganism producing theenzyme, more particularly an intact recombinant microorganism producingthe enzyme.

In an embodiment of the invention, the cyclization reaction is carriedout in the presence of an SHC biocatalyst capable of bioconvertinghomofarnesol to (−)-Ambrox.

The SHC biocatalyst is a wild-type or a variant enzyme or is amicroorganism expressing a gene encoding the SHC enzyme, preferably arecombinant E. coli microorganism. The SHC biocatalyst can be used inany form such as but not limited to a purified SHC enzyme, a crudeextract containing an SHC enzyme or an immobilised SHC enzyme (e.g. on acarrier), or the biocatalyst can be a microorganism having produced orproducing the SHC enzyme, such as an intact recombinant whole celland/or fragmented cell or a membrane fraction containing the SHC enzyme.

In a particular embodiment of the present invention, the homofarnesolmixture is enriched in the 7E,3E-geometric isomer.

In a more particular embodiment, the homofarnesol mixture is at least55/45 by weight 7E,3E/7E,3Z.

In a more particular embodiment, the homofarnesol mixture is at least70/30 by weight 7E,3E/7E,3Z.

In a still more particular embodiment, the homofarnesol mixture is atleast 80/20 by weight 7E,3E/7E,3Z

In a still more particular embodiment, the homofarnesol mixture is atleast 90/10 by weight 7E,3E/7E,3Z.

In a still more particular embodiment, the homofarnesol mixture is atleast 95/5 by weight 7E,3E/7E,3Z.

In a particular embodiment of the present invention, the homofarnesolmixture consists of 7E,3E/Z-geometric isomers and no other geometricisomers of homofarnesol.

The skilled person understands that the term 7E, 7Z, 3E or 3Z used inconnection with homofarnesol refers respectively to the orientation ofthe double bond at the 7-position and 3-position of homofarnesol. The7E,3E-homofarnesol compound has the CAS No. 459-89-2, whereas the7E,3Z-homofarnesol compound has the CAS No. 138152-06-4. The use of theterm 7E, 3E/Z-homofarnesol refers to a mixture of the compounds.

Methods of obtaining homofarnesol mixtures useful as a substrate in thecyclisation reaction in accordance with the method of the presentinvention are set forth in the co-pending applications PCT/EP2014/072891(published as WO2015/059293) and PCT2014/EP/072882 (published asWO2015/059290) referred to above, which are hereby incorporated byreference in their entirety. In general terms, they describe a synthesisof homofarnesol mixtures that proceeds by converting farnesene, moreparticularly alpha-farnesene and/or beta-farnesene, to its correspondingcyclopropanated farnesene derivative, using an organic solution of anN-alkyl-N-nitroso urea. The cyclopropanated derivative then undergoesring-opening and rearrangement reactions in the presence of a Bronstedacid to afford the homofarnesol mixture, which is selective for the7E,3E geometric isomer. Using farnesene, as a starting material isparticularly preferred because it ensures that the E-configuration ofthe double bond at the 7 position of homofarnesol is fixed.

Specific reaction conditions, which form particular embodiments of thepresent invention, are set forth in the co-pending applications, as wellas the examples hereinbelow, and do not require more elaboration here.

The cyclization of homofarnesol to provide a reaction mixture containing(−)-Ambrox may be catalysed by Squalene Hopene Cyclase (SHC). SHC may bea wild type enzyme (e.g. SEQ ID No. 1), or a variant thereof (e.g. SEQID No. 2, or SEQ ID No. 4). SHC can be obtained from Alicyclobacillusacidocaldarius (Bacillus acidocaldarius), Zymomonas mobilis orBradyrhizobium japonicum (as set forth in Example 3b ofUS20120135477A1).

However, the enzyme can also be produced by recombinant means, usingtechniques that are generally known in the art.

The term “recombinant” as used with respect to the production of enzymesshall refer to enzymes produced by recombinant DNA techniques, i.e.,produced from cells transformed by an exogenous DNA construct encodingthe desired enzyme. The term “recombinant DNA” therefore includes arecombinant DNA incorporated into a vector into an autonomouslyreplicating plasmid or virus, or into the genomic DNA of a prokaryote oreukaryote (or the genome of a homologous cell, at a position other thanthe natural chromosomal location).

Nucleic acid molecule(s) is/are operatively linked to expression controlsequences allowing expression in prokaryotic and/or eukaryotic hostcells. As used herein, “operatively linked” means incorporated into agenetic construct so that expression control sequences effectivelycontrol expression of a coding sequence of interest. Thetranscriptional/translational regulatory elements referred to aboveinclude but are not limited to inducible and non-inducible,constitutive, cell cycle regulated, metabolically regulated promoters,enhancers, operators, silencers, repressors and other elements that areknown to those skilled in the art and that drive or otherwise regulategene expression. Such regulatory elements include but are not limited toregulatory elements directing constitutive expression or which allowinducible expression like, for example, CUP-1 promoter, thetet-repressor as employed, for example, in the tet-on or tet-offsystems, the lac system, the trp system regulatory elements. By way ofexample, Isopropyl β-D-1-thiogalactopyranoside (IPTG) is an effectiveinducer of protein expression in the concentration range of 100 μM to1.0 mM. This compound is a molecular mimic of allolactose, a lactosemetabolite that triggers transcription of the lac operon, and it istherefore used to induce protein expression where the gene is under thecontrol of the lac operator.

Similarly, nucleic acid molecule(s) can form part of a hybrid geneencoding additional polypeptide sequences, for example, a sequence thatfunctions as a marker or reporter. Examples of marker and reporter genesincluding beta-lactamase, chloramphenicol acetyltransferase (CAT),adenosine deaminase (ADA), aminoglycoside phosphotransferasedihydrofolate reductase (DHFR), hygromycin-B-phosphotransferase (HPH),thymidine kinase (TK), lacZ (encoding beta-galactosidase), and xanthineguanine phosphoribosyltransferase (XGPRT). As with many of the standardprocedures associated with the practice of the disclosure, skilledartisans will be aware of additional useful reagents, for example,additional sequences that can serve the function of a marker orreporter.

Recombinant polynucleotides can encode SHC enzymes such as the wild typeSHC or a variant thereof, which may be inserted into a vector forexpression and optional purification. One type of vector is a plasmidrepresenting a circular double stranded DNA loop into which additionalDNA segments are ligated. Certain vectors can control the expression ofgenes to which they are functionally linked. These vectors are called“expression vectors”. Usually expression vectors suitable for DNArecombination techniques are of the plasmid type. Typically, anexpression vector comprises a gene such as the wild type SHC or avariant thereof. In the present description, the terms “plasmid” and“vector” are used interchangeably since the plasmid is the vector typemost often used.

Such vectors can include DNA sequences which include but are not limitedto DNA sequences that are not naturally present in the host cell, DNAsequences that are not normally transcribed into RNA or translated intoa protein (“expressed”) and other genes or DNA sequences which onedesires to introduce into the non-recombinant host. It will beappreciated that typically the genome of a recombinant host is augmentedthrough the stable introduction of one or more recombinant genes.However, autonomous or replicative plasmids or vectors can also be usedwithin the scope of this disclosure. Moreover, the present disclosurecan be practiced using a low copy number, e.g., a single copy, or highcopy number plasmid or vector.

In a preferred embodiment the vector of the present disclosure comprisesplasmids, phagemids, phages, cosmids, artificial bacterial andartificial yeast chromosomes, knock-out or knock-in constructs.Synthetic nucleic acid sequences or cassettes and subsets may beproduced in the form of linear polynucleotides, plasmids, megaplasmids,synthetic or artificial chromosomes, such as plant, bacterial, mammalianor yeast artificial chromosomes.

It is preferred that the proteins encoded by the introducedpolynucleotide are produced within the cell upon introduction of thevector. The diverse gene substrates may be incorporated into plasmids.The plasmids are often standard cloning vectors, e.g., bacterialmulticopy plasmids. The substrates can be incorporated into the same ordifferent plasmids. Often at least two different types of plasmid havingdifferent types of selectable markers are used to allow selection forcells containing at least two types of vectors.

Typically bacterial or yeast cells may be transformed with any one ormore of the following nucleotide sequences as is well known in the art.For in vivo recombination, the gene to be recombined with the genome orother genes is used to transform the host using standard transformingtechniques. In a suitable embodiment DNA providing an origin ofreplication is included in the construct. The origin of replication maybe suitably selected by the skilled person. Depending on the nature ofthe genes, a supplemental origin of replication may not be required ifsequences are already present with the genes or genome that are operableas origins of replication themselves.

A bacterial or yeast cell may be transformed by exogenous orheterologous DNA when such DNA has been introduced inside the cell. Thetransforming DNA may or may not be integrated, i.e. covalently linkedinto the genome of the cell. In prokaryotes, and yeast, for example, thetransforming DNA may be maintained on an episomal element such as aplasmid. With respect to eukaryotic cells, a stably transformed cell isone in which the transfected DNA has become integrated into a chromosomeso that it is inherited by daughter cells through chromosomereplication. This stability is demonstrated by the ability of theeukaryotic cell to establish cell lines or clones comprised of apopulation of daughter cells containing the transforming DNA.

Generally, the introduced DNA is not originally resident in the hostthat is the recipient of the DNA, but it is within the scope of thedisclosure to isolate a DNA segment from a given host, and tosubsequently introduce one or more additional copies of that DNA intothe same host, e.g., to enhance production of the product of a gene oralter the expression pattern of a gene. In some instances, theintroduced DNA will modify or even replace an endogenous gene or DNAsequence by, e.g., homologous recombination or site-directedmutagenesis. Suitable recombinant hosts include microorganisms, plantcells, and plants.

The present disclosure also features recombinant hosts. The term“recombinant host”, also referred to as a “genetically modified hostcell” or a “transgenic cell” denotes a host cell that comprises aheterologous nucleic acid or the genome of which has been augmented byat least one incorporated DNA sequence. A host cell of the presentdisclosure may be genetically engineered with the polynucleotide or thevector as outlined above.

The host cells that may be used for purposes of the disclosure includebut are not limited to prokaryotic cells such as bacteria (for example,E. coli and B. subtilis), which can be transformed with, for example,recombinant bacteriophage DNA, plasmid DNA, bacterial artificialchromosome, or cosmid DNA expression vectors containing thepolynucleotide molecules of the disclosure; simple eukaryotic cells likeyeast (for example, Saccharomyces and Pichia), which can be transformedwith, for example, recombinant yeast expression vectors containing thepolynucleotide molecule of the disclosure. Depending on the host celland the respective vector used to introduce the polynucleotide of thedisclosure the polynucleotide can integrate, for example, into thechromosome or the mitochondrial DNA or can be maintainedextrachromosomally like, for example, episomally or can be onlytransiently comprised in the cells.

The term “cell” as used herein in particular with reference to geneticengineering and introducing one or more genes or an assembled cluster ofgenes into a cell, or a production cell is understood to refer to anyprokaryotic or eukaryotic cell. Prokaryotic and eukaryotic host cellsare both contemplated for use according to the disclosure, includingbacterial host cells like E. coli or Bacillus sp, yeast host cells, suchas S. cerevisiae, insect host cells, such as Spodoptora frugiperda orhuman host cells, such as HeLa and Jurkat.

Specifically, the cell is a eukaryotic cell, preferably a fungal,mammalian or plant cell, or prokaryotic cell. Suitable eucaryotic cellsinclude, for example, without limitation, mammalian cells, yeast cells,or insect cells (including Sf9), amphibian cells (including melanophorecells), or worm cells including cells of Caenorhabditis (includingCaenorhabditis elegans). Suitable mammalian cells include, for example,without limitation, COS cells (including Cos-1 and Cos-7), CHO cells,HEK293 cells, HEK293T cells, HEK293 T-Rex™ cells, or other transfectableeucaryotic cell lines. Suitable bacterial cells include withoutlimitation E. coli.

Preferably prokaryotes, such as E. coli, Bacillus, Streptomyces, ormammalian cells, like HeLa cells or Jurkat cells, or plant cells, likeArabidopsis, may be used.

Preferably the cell is an Aspergillus sp or a fungal cell, preferably,it can be selected from the group consisting of the generaSaccharomyces, Candida, Kluyveromyces, Hansenula, Schizosaccharomyces,Yarrowia, Pichia and Aspergillus.

Preferably the E. coli host cell is an E. coli host cell which isrecognized by the industry and regulatory authorities (including but notlimited to an E. coli K12 host cell or as demonstrated in the Examples,an E. coli BL21 host cell).

One preferred host cell to use with the present disclosure is E. coli,which may be recombinantly prepared as described herein. Thus, therecombinant host may be a recombinant E. coli host cell. There arelibraries of mutants, plasmids, detailed computer models of metabolismand other information available for E. coli, allowing for rationaldesign of various modules to enhance product yield. Methods similar tothose described above for Saccharomyces can be used to make recombinantE. coli microorganisms.

In one embodiment, the recombinant E. coli microorganism comprisesnucleotide sequences encoding SHC genes or functionalequivalents/homologies thereof including but not limited to variants,homologues mutants, derivatives or fragments thereof.

Another preferred host cell to use with the present disclosure is S.cerevisiae which is a widely used chassis organism in synthetic biology.Thus, the recombinant host may be S. cerevisiae. There are libraries ofmutants, plasmids, detailed computer models of metabolism and otherinformation available for S. cerevisiae, allowing for rational design ofvarious modules to enhance product yield. Methods are known for makingrecombinant S. cerevisiae microorganisms.

Culturing of cells is performed, in a conventional manner. The culturemedium contains a carbon source, at least one nitrogen source andinorganic salts, and vitamins are added to it. The constituents of thismedium can be the ones which are conventionally used for culturing thespecies of microorganism in question.

Carbon sources of use in the instant method include any molecule thatcan be metabolized by the recombinant host cell to facilitate growthand/or production of (−)-Ambrox. Examples of suitable carbon sourcesinclude, but are not limited to, sucrose (e.g., as found in molasses),fructose, xylose, glycerol, glucose, cellulose, starch, cellobiose orother glucose containing polymer.

In embodiments employing yeast as a host, for example, carbon sourcessuch as sucrose, fructose, xylose, ethanol, glycerol, and glucose aresuitable. The carbon source can be provided to the host organismthroughout the cultivation period or alternatively, the organism can begrown for a period of time in the presence of another energy source,e.g., protein, and then provided with a source of carbon only during thefed-batch phase.

The suitability of a recombinant host cell microorganism for use in themethods of the present disclosure may be determined by simple testprocedures using well known methods. For example, the microorganism tobe tested may be propagated in a rich medium (e.g., LB-medium,Bacto-tryptone yeast extract medium, nutrient medium and the like) at apH, temperature and under aeration conditions commonly used forpropagation of the microorganism. Once recombinant microorganisms (i.e.recombinant host cells) are selected that produce the desired productsof bioconversion, the products are typically produced by a productionhost cell line on the large scale by suitable expression systems andfermentations, e.g. by microbial production in cell culture.

In one embodiment of the present disclosure, a defined minimal mediumsuch as M9A is used for cell cultivation. The components of M9A mediumcomprise: 14 g/L KH₂PO₄, 16 g/L K₂HPO₄, 1 g/L Na₃Citrate.2H₂O, 7.5 g/L(NH₄)₂SO₄, 0.25 g/L MgSO₄.7H₂O, 0.015 g/L CaCl₂.2H₂O, 5 g/L of glucoseand 1.25 g/L yeast extract).

In another embodiment of the present disclosure, nutrient rich mediumsuch as LB (Luria-Bertani) was used. The components of LB comprise: 10g/L tryptone, 5 g/L yeast extract, 5 g/L NaCl).

Other examples of Mineral Medium and M9 Mineral Medium are disclosed,for example, in U.S. Pat. No. 6,524,831B2 and US 2003/0092143A1.

The recombinant microorganism may be grown in a batch, fed batch orcontinuous process or combinations thereof. Typically, the recombinantmicroorganism is grown in a fermentor at a defined temperature in thepresence of a suitable nutrient source, e.g., a carbon source, for adesired period of time to bioconvert homofarnesol to (−)-Ambrox in adesired amount.

The recombinant host cells may be cultivated in any suitable manner, forexample by batch cultivation or fed-batch cultivation. As used herein,the term “batch cultivation” is a cultivation method in which culturemedium is neither added nor withdrawn during the cultivation. As usedherein, the term “fed-batch” means a cultivation method in which culturemedium is added during the cultivation but no culture medium iswithdrawn.

One embodiment of the present disclosure provides a method of producing(−)-Ambrox in a cellular system comprising producing wild type SHC orvariants thereof under suitable conditions in a cellular system, feedinghomofarnesol to the cellular system, converting the homofarnesol to(−)-Ambrox using the SHC or variants produced using the cellular system,collecting (−)-Ambrox from cellular system and isolating the (−)-Ambroxfrom the system. Expression of other nucleotide sequences may serve toenhance the method. The bioconversion method can include the additionalexpression of other nucleotide sequences in the cellular system. Theexpression of other nucleotide sequences may enhance the bioconversionpathway for making (−)-Ambrox.

A further embodiment of the present disclosure is a bioconversion methodof making (−)-Ambrox comprising growing host cells comprising wild typeSHC or variant genes, producing wild type SHC or variant enzymes in thehost cells, feeding homofarnesol (e.g. EEH) to the host cells,incubating the host cells under conditions of pH, temperature andsolubilizing agent suitable to promote the conversion of homofarnesol toAmbrox and collecting (−)-Ambrox. The production of the wild type SHC orvariant enzymes in the host cells provides a method of making (−)-Ambroxwhen homofarnesol is added to the host cells under suitable reactionconditions. Achieved conversion may be enhanced by adding morebiocatalyst and SDS to the reaction mixture.

The recombinant host cell microorganism may be cultured in a number ofways in order to provide cells in suitable amounts expressing the wildtype SHC or variant enzymes for the subsequent bioconversion step. Sincethe microorganisms applicable for the bioconversion step vary broadly(e.g. yeasts, bacteria and fungi), culturing conditions are, of course,adjusted to the specific requirements of each species and theseconditions are well known and documented. Any of the art known methodsfor growing cells of recombinant host cell microorganisms may be used toproduce the cells utilizable in the subsequent bioconversion step of thepresent disclosure. Typically the cells are grown to a particulardensity (measurably as optical density (OD)) to produce a sufficientbiomass for the bioconversion reaction. The cultivation conditionschosen influence not only the amount of cells obtained (the biomass) butthe quality of the cultivation conditions also influences how thebiomass becomes a biocatalyst. The recombinant host cell microorganismexpressing the wild type SHC or variant genes and producing the wildtype SHC or variant enzymes is termed a biocatalyst which is suitablefor use in a bioconversion reaction. In some embodiments the biocatalystis a recombinant whole cell producing wild type SHC or variant enzymesor it may be in suspension or an immobilized format.

In one embodiment, the biocatalyst is produced in sufficient amounts (tocreate a sufficient biomass), harvested and washed (and optionallystored (e.g. frozen or lyophilized)) before the bioconversion step.

In a further embodiment, the cells are produced in sufficient amounts(to create a sufficient biocatalyst) and the reaction conditions arethen adjusted without the need to harvest and wash the biocatalyst forthe bioconversion reaction. This one step (or “one pot”) method isadvantageous as it simplifies the process while reducing costs. Theculture medium used to grow the cells is also suitable for use in thebioconversion reaction provided that the reaction conditions areadjusted to facilitate the bioconversion reaction.

The bioconversion methods of the present disclosure are carried outunder conditions of time, temperature, pH and solubilizing agent toprovide for conversion of the homofarnesol feedstock to (−)-Ambrox ThepH of the reaction mixture may be in the range of 4-8, preferably, 5 to6.5, more preferably 4.8-6.0 for the SHC variant enzymes and in therange of from about pH 5.0 to about pH 7.0 for the wild type SHC enzymeand can be maintained by the addition of buffers to the reactionmixture. An exemplary buffer for this purpose is a citric acid buffer.The preferred temperature is between from about 15° C. and about 45° C.,preferably about 20° C. and about 40° C. although it can be higher, upto 55° C. for thermophilic organisms especially if the wild type enzymefrom a thermophilic microorganism is used. The temperature can be keptconstant or can be altered during the bioconversion process.

The Applicant has demonstrated that it may be useful to include asolubilizing agent (e.g. a surfactant, detergent, solubility enhancer,water miscible organic solvent and the like) in the bioconversionreaction. Examples of surfactants include but are not limited to TritonX-100, Tween 80, taurodeoxycholate, Sodium taurodeoxycholate, Sodiumdodecyl sulfate (SDS), and/or sodium lauryl sulfate (SLS).

The Applicant has selected and identified SDS as a particularly usefulsolubilizing agent from a long list of other less useful solubilizingagents. In particular, the Applicant identified SDS as a remarkablybetter solubilizing agent than e.g. Triton X-100 in terms of reactionvelocity and yield for the homofarnesol to (−)-Ambrox bioconversionreaction.

Without wishing to be bound by theory, the use of SDS with recombinantmicrobial host cells may be advantageous as the SDS may interactadvantageously with the host cell membrane in order to make the SHCenzyme (which is a membrane bound enzyme) more accessible to thehomofarnesol substrate. In addition, the inclusion of SDS at a suitablelevel in the reaction mixture may improve the properties of the emulsion(homofarnesol in water) and/or improve the access of the homofarnesolsubstrate to the SHC enzyme within the host cell while at the same timepreventing the disruption (e.g. denaturation/inactivation of the wildtype SHC or variant enzyme).

The concentration of the solubilising agent (e.g. SDS) used in thebioconversion reaction is influenced by the biomass amount and thesubstrate (EEH) concentration. That is, there is a degree ofinterdependency between the solubilising agent (e.g. SDS) concentration,the biomass amount and the substrate (EEH) concentration. By way ofexample, as the concentration of homofarnesol substrate increases,sufficient amounts of biocatalyst and solubilising agent (e.g. SDS) arerequired for an efficient bioconversion reaction to take place. If, forexample, the solubilising agent (e.g. SDS) concentration is too low, asuboptimal homofarnesol conversion may be observed. On the other hand,if, for example, the solubilising agent (e.g. SDS) concentration is toohigh, then there may be a risk that the biocatalyst is affected througheither the disruption of the intact microbial cell and/or andenaturation/inactivation of the SHC/HAC enzyme.

The selection of a suitable concentration of SDS in the context of thebiomass amount and substrate (EEH) concentration is within the knowledgeof the Skilled Person. By way of example, a predictive model isavailable to the Skilled Person to determine the suitable SDS, substrate(EEH) and biomass concentrations.

The temperature of the bioconversion reaction for a wild type SHC enzymeis from about 45-60° C., preferably 55° C.

The pH range of the bioconversion reaction for a wild type SHC enzyme isfrom about 5.0 to 7.0, more preferably from about 5.6 to about 6.2, evenmore preferably about 6.0.

The temperature of the bioconversion reaction for a SHC variant enzymeis about 34° C. to about 50° C., preferably about 35° C.

The pH of the bioconversion reaction for a SHC variant enzyme is about4.8-6.4, preferably about 5.2-6.0.

Preferably the solubilising agent used in the bioconversion reaction isSDS.

The [SDS]/[cells] ratio is in the range of about, 10:1-20:1, preferablyabout 15:1-18:1, preferably about 16:1 when the ratio of biocatalyst toEEH homofarnesol is about 2:1

The SDS concentration in the bioconversion reaction for a SHC variantenzyme is in the range of about 1-2%, preferably in the range of about1.4-1.7%, even more preferably about 1.5% when the homofarnesolconcentration is about 125 g/l EEH and the biocatalyst concentration is250 g/l (corresponding to an OD of about 175 (650 nm)).

The ratio of biocatalyst to EEH homofarnesol substrate is in the rangeof about 0.5:1-2:1, in some embodiments 2:1, preferably about 1:1 or0.5:1.

In some embodiments, (−)-Ambrox is produced using a biocatalyst to whichthe homofarnesol substrate is added. It is possible to add the substrateby feeding using known means (e.g. peristaltic pump, infusion syringeand the like). Homofarnesol is an oil soluble compound and is providedin an oil format. Given that the biocatalyst is present in an aqueousphase, the bioconversion reaction may be regarded as a two phase systemwhen homofarnesol is added to the bioconversion reaction mixture. Thisis the case even when a solubilizing agent (e.g. SDS) is present.

Further details of a suitable bioconversion process are disclosed in theexamples, set forth herein below.

The bioconversion process according to the present invention produces areaction mixture containing the desired (−)-Ambrox, and also a number ofby-products. More particularly, the reaction mixture contains, inaddition to (−)-Ambrox a complex mixture of by-products, including anovel constitutional isomer of (−)-Ambrox according to the formula (II),as well as known stereo isomers of (−)-Ambrox according to the formulae(III) and (IV)

The applicant believes, although does not intend to be bound by anyparticular theory, that the compound of formula (II) is formed by thecyclization of the 7E,3Z-geometric isomer of homofarnesol. It has beendescribed as practically odourless, with a detection threshold of >500ng/l.

As stated above, the applicant believes that the compound of formula(II) is a novel molecule, and as such, this compound forms a furtheraspect of the present invention. Perfume ingredients and perfumecompositions consisting of or comprising the compound (II), as well asperfumed articles containing same, form additional aspects of theinvention.

The use of the compound of formula (II) as a perfume ingredient inperfumery applications, such as fine perfumes or functional perfumecompositions such as personal care, household care and fabric carecompositions, forms further additional aspects of the invention.

Mixtures of (−)-Ambrox and an olfactory acceptable amount of compound(II) forms still another aspect of the present invention.

The term “olfactory acceptable amount” as used herein in relation to thecompound of formula (II), or any of the other by-products (III) or (IV),or indeed, any material that may be present as an impurity in (−)-Ambroxformed in accordance with a method of the present invention, isunderstood to mean that the compound or material is present in a mixturewith (−)-Ambrox in an amount below its odour detection threshold, or inan amount at which it will not contribute its olfactory characteristicsin a way that will affect the olfactory character of (−)-Ambrox.(−)-Ambrox containing an olfactory acceptable amount of any suchcompound or material would be identifiable to a skilled perfumer aspossessing the odour character of commercial grades of (−)-Ambrox, suchas AMBROFIX™ obtained by a synthetic procedure ex-sclareol, andavailable from Givaudan.

In preferred embodiments of the present invention, the reaction mixturecontains no, or substantially no, unreacted homofarnesol.

The applicant discovered that homofarnesol was a powerful solvent for(−)-Ambrox as well as for the aforementioned by-products of thebioconversion process. As such, in the presence of appreciable amountsof homofarnesol, (−)-Ambrox and the by-products remain dissolvedtogether in a crude, intractable mixture, from which it is difficult,protracted and costly to separate and ultimately isolate (−)-Ambrox inolfactively pure form. Reducing the level of unreacted homofarnesol inadmixture with (−)-Ambrox and the compounds (II), (III) and (IV) wasfound to considerably facilitate downstream processing andisolation/purification of (−)-Ambrox.

Downstream processing, as will be appreciated by persons skilled in theart, is a critical operation in the manufacture of useful compoundsformed by bioconversion processes. As part of the synthesis of acompound, it can affect the compound's physical properties. In the caseof the preparation of perfume ingredients by biotech methods, it isdesirable that a target compound can be separated from a reactionmixture in olfactively pure form in order that the desired odourcharacteristics of the target compound are not distorted by odourcontributions of the complex mixture of contaminants and by-productsthat may be present in the fermentation medium or the biocatalyst.

Accordingly, the invention provides in another of its aspects a methodof isolating and purifying (−)-Ambrox from a reaction mixture,comprising one or more of the compounds (II), (III) and (IV).

In yet another aspect of the present invention there is provided amethod of improving or enhancing the odour of (−)-Ambrox, comprising thesteps of separation and purification of (−)-Ambrox from a reactionmixture containing one or more of the compounds (II), (III) and (IV).

In an isolated and purified form, (−)-Ambrox either does not contain anyof the compounds (II), (III) or (IV), or if it does contain any of saidcompounds, then each should be present in an olfactory acceptableamount.

The reaction mixture obtained from the bioconversion process, such as aprocess as described herein above, generally comprises a solid phasecontaining crude (−)-Ambrox and one or more of the by-products (II),(III) and (IV), as well as cellular material and/or debris thereof; anda liquid phase or liquid phases comprising water and/or any unreactedhomofarnesol.

The solid phase may be separated from the liquid phase or phases byfiltration or centrifugation. Furthermore, by selecting a filter with anappropriate pore size, it is also possible to effect separation of thecrude (−)-Ambrox, from the cellular material and/or debris. Once thecrude (−)-Ambrox is separated from cellular material and/or debristhereof, it may be washed, before being subjected to further work-upprocedures to isolate (−)-Ambrox from compounds (II), (III) and (IV).

Alternatively, instead of filtration or centrifugation, the reactionmixture can be warmed to a temperature above the melting point of(−)-Ambrox, whereupon (−)-Ambrox forms an oil phase above an aqueousphase containing the cellular material and debris. Optionally, and inorder to ensure a complete recovery of (−)-Ambrox, the aqueous andcellular material can be washed with a water-immiscible organic solvent(such as toluene) to remove any residual (−)-Ambrox that may have beenentrained in the aqueous phase, and these washings can be combined withthe oil phase. The oil phase can thereafter be concentrated byevaporation to provide a crude mixture comprising (−)-Ambrox and one ormore of the compounds (II), (III) and (IV),which mixture can thensubjected to further work-up procedures to isolate and purify(−)-Ambrox.

In another embodiment, instead of warming the reaction mixture to form a(−)-Ambrox-containing oil phase, the reaction mixture can be extractedwith a suitable water-immiscible organic solvent (such as toluene) toform an organic phase containing (−)-Ambrox and one or more of thecompounds (II), (III) and (IV), which can be separated from an aqueousphase containing the cellular material and debris. The organic phase canbe concentrated by evaporation to provide a crude mixture comprising(−)-Ambrox and one or more of the compounds (II), (III) and (IV), whichcan be subjected to further work-up procedures to isolate and purify(−)-Ambrox.

In yet another alternative method, the reaction mixture can be steamdistilled to remove the distillate from the cellular material anddebris. The distillate can be collected as a biphasic mixture, beforethe oil phase of the biphasic mixture comprising a mixture of (−)-Ambroxand one or more of the compounds (II), (III) and (IV) is separated fromthe aqueous phase, and then subjected to further work-up procedures toisolate and purify (−)-Ambrox.

In a particular embodiment of the present invention, said method ofisolating and purifying (−)-Ambrox comprises the step of selectivelycrystallizing (−)-Ambrox from a mixture containing one or more of thecompounds (II), (III) or (IV).

The phrase “selectively crystallizing” refers to a process step whereby(−)-Ambrox is caused to crystallize from a solvent, whilst the compounds(II), (III) and (IV) remain dissolved in the crystallizing solvent, tosuch an extent that isolated crystalline material contains only(−)-Ambrox, or if it contains any of the compounds (II), (III) or (IV),then they are present only in olfactory acceptable amounts.

Crystallization may be carried out in a suitable organic solvent. Thechoice of solvent is based on considerations, such as solubilitydifferences at room temperature and at high temperatures, or in boilingsolvent; and for the need of an abundance of crystals recoverable incool solvent. Usually, a compound to be separated is dissolved in arelatively polar solvent and then a relatively less polar solvent can beadded to bring the dissolved compound to its solubility limit, whereuponit will start to crystallize. Also, in an industrial process, issues ofcost as well as safety of handling are relevant. Suitable solventsinclude, but are not limited to methanol, acetone, petroleum ether,hexane, t-butyl methyl ether, THF and ethyl acetate. Preferred solventsinclude toluene or ethyl alcohol. Pairs of solvents may also beemployed.

In a particularly preferred embodiment of the present invention,selective crystallization is undertaken by dissolving the mixturecontaining (−)-Ambrox and one or more of the compounds (II), (III) and(IV) in warm ethanol and allowing (−)-Ambrox to selectively crystallizeby slowly adding a non-solvent, such as water, to the cooling ethanolicsolution.

Considering the close structural relationship of (−)-Ambrox and theby-product compounds (II), (III) and (IV), which are respectively aconstitutional isomer and two stereoisomers of (−)-Ambrox, it wasremarkable that (−)-Ambrox could be selectively crystallized from such amixture, to provide (−)-Ambrox in olfactively pure form and in highyields. The skilled person would reasonably anticipate that thecompounds would co-crystallize with (−)-Ambrox, rendering downstreamprocessing far more complex, time-consuming and expensive than was foundto be the case.

The surprisingly facile manner in which (−)-Ambrox could be separatedfrom a mixture containing compound (II), (III) and/or (IV) bycrystallization represents a particular advantage of the presentinvention.

The ease with which (−)-Ambrox could be separated by crystallizationcould be contrasted with the observation that (−)-Ambrox could not berecovered in such a facile manner and in such high yield from a mixturecontaining (II), (III) and/or (IV) by other purification techniques,such as by distillation, owing to the very similar boiling points of(−)-Ambrox and the by-products (II), (III) and (IV).

The term “olfactively pure” as it is used in relation to (−)-Ambrox, isintended to mean that (−)-Ambrox is free of compounds (II), (III) or(IV), or any other material found in the reaction mixture, or that ifsuch compounds or materials should be present, they are present inolfactory acceptable amounts, as that term is defined herein.

In an embodiment of the invention (−)-Ambrox in olfactively pure formcontains less than 5% by weight of any of the compounds (II), (III) or(IV).

In more particular embodiments, (−)-Ambrox in olfactively pure formcontains less than 4%, less than 3%, less than 2%, less than 1%, lessthan 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than0.5%, less than 0.4%, less than 0.3%, less than 0.2%, less than 0.1%, orless than 0.05% by weight of each of the compounds (II), (III) or (IV).

The quality of separation of (−)-Ambrox from the mixture of thecompounds (II), (III) and/or (IV) by selective crystallization may beinfluenced by the composition of the mixture from which it is separated.More particularly, the quality of the separation of (−)-Ambrox from amixture of compounds (II), (III) and/or (IV) by crystallization wasimproved when the weight ratio of (−)-Ambrox to the other compounds(II), (III) and (IV) in the mixture was greater than 70:30, moreparticularly 80:20, more particularly 90:10, still more particularly95:5, and more particularly still 97:3.

Furthermore, the quality of the separation of (−)-Ambrox bycrystallization may be influenced by the amount of unreactedhomofarnesol present in the mixture from which it is separated. Moreparticularly, the quality of separation is improved when the level ofunreacted homofarnesol is less than 30 wt % by weight, more particularlyless than 20 wt %, more particularly less than 10% by weight, moreparticularly still less than 5 wt % and still more particularly lessthan 3% by weight, still more particularly less than 2% by weight, andmore particularly still less than 1% by weight, based on the weight ofthe mixture from which (−)-Ambrox is crystallized.

Preferably, the reagents and reaction conditions employed in thebioconversion process of the present invention are such that thereaction proceeds with 100% conversion of homofarnesol, or substantiallyso, thus leaving no unreacted homofarnesol in the reaction mixture.However, if unreacted homofarnesol is present, although economicallydisadvantageous, it can be separated from (−)-Ambrox and otherby-products by distillation, for example.

Accordingly, in a particular embodiment of the invention, there isprovided a method of isolating and purifying (−)-Ambrox from a mixturecomprising one or more of the compounds (II), (III) and (IV), whichmixture is free, or substantially free, of homofarnesol.

In a more particular embodiment, the isolation and purification of(−)-Ambrox from a mixture comprising one or more of the compounds (II),(III) and (IV), and free or substantially free of homofarnesol, isachieved by the selective crystallization of (−)-Ambrox.

(−)-Ambrox obtained according to a method of the present invention isobtained in olfactively pure form. Olfactively pure (−)-Ambrox formsanother aspect of the present invention.

(−)-Ambrox in crystalline form forms yet another aspect of the presentinvention.

(−)-Ambrox formed in accordance with the method of the present inventionmay be mixed with one or more additional perfume ingredients to formperfume compositions that find use in perfumery applications, includinguse in fine perfumery, as well as use in consumer products, such aspersonal care, fabric care and household care applications.

Accordingly, the invention provides in another of its aspects a perfumecomposition comprising (−)-Ambrox and at least one other perfumeingredient, wherein said perfume composition contains olfactoryacceptable amounts of one or more of the compounds (II), (III) or (IV).

The invention will be further illustrated with reference to thefollowing examples.

EXAMPLES Example 1

Preparation of Homofarnesol

General Analytical Conditions:

Non-polar GC/MS: 50° C./2 min, 20° C./min 200° C., 35° C./min 270° C.GC/MS Agilent 5975C MSD with HP 7890A Series GC system. Non-polarcolumn: BPX5 from SGE, 5% phenyl 95% dimethylpolysiloxane 0.22 mm×0.25mm×12 m. Carrier Gas: Helium. Injector temperature: 230° C. Split 1:50.Flow: 1.0 ml/min. Transfer line: 250° C. MS-quadrupol: 106° C.MS-source: 230° C.

A) Preparation of MNU in THF

A solution of urea (175 g, 2.9 mol) and methylamine hydrochloride (198g, 2.9 mol) in water (400 ml) is heated at reflux (105° C.) for 3.5 hunder stirring. At 40° C. NaNO2 (101 g, 1.45 mol) dissolved in water(200 ml) is added. After 15 min THF (1000 ml) is added which results ina transparent 2-phase mixture. Conc. H2SO4 (110 g, 1.1 mol) is added at0-5° C. and stirring within 1.5 h. After another 0.5 h at 0-5° C. thetwo transparent phases are separated at 25° C. The organic phase (A)(1065 ml, theoretically 1.35 M) is stored for a few days at 0-5° C. orforwarded immediately to the cyclopropanation reactor.

After phase separation the water phase is extracted twice with THF (2×1l). This gives 1100 ml of phase B and 1075 of phase C. Whereas phase Agives a 51% conversion of a terminal alkene to a cyclopropane in asubsequent cyclopropanation reaction, phase B gives <0.5% cyclopropaneand phase C gives no detectable conversion. We conclude that >99% MNU isextracted after the first phase separation. Usually the water phase istherefore discarded after the first phase separation (from organic phaseA) after treatment with conc. aqueous KOH and acetic acid.

B) Preparation of E-Δ-Farnesene Using MNU in THF

N-Methyl-N-nitroso urea 1.35 M in THF (136 ml, 184 mmol) is addeddropwise at 0° C. to a rapidly stirred mixture of E-beta-Farnesene (CAS18794-84-8) (25 g, 122 mmol) and aqueous KOH (50 ml, 40%) at 0-5° C.After the addition of 4 ml of the MNU solution, Pd(acac)2 (7.4 mg, 0.024mmol, 0.02%) pre-dissolved in 0.5 ml dichloromethane is added. Theremaining MNU solution is added over 4 h at 0-5° C. A GC at this stageshowed 28% unconverted E-beta-Farnesene, 65% of the desiredmonocyclopropane (shown above) and 3% of a biscyclopropanated compound5. After 16 h at 25° C. acetic acid (100 ml) is added at 0-5° C., thentert-butyl methyl ether (250 ml). After phase separation the organicphase is washed with 2M HCl (250 ml) and the aqueous phase extractedwith tert-butyl methyl ether (250 ml). The combined organic layers arewashed with water (2×100 ml), aqueous 10% NaOH (2×100 ml) and water(2×100 ml), dried over MgSO₄, filtered and concentrated to give 26.9 gof a slightly yellow liquid which contains 9% E-beta-Farnesene, 82% ofthe desired monocyclopropane compound and 6% of a biscyclopropanatedside product.

The desired compound could be further isolated by distillativepurification.

Addition of 1 g K₂CO₃ (1 g) and distillation over a 30 cm steel coilcolumn at 40-60 mbar gives 147 g monocyclopropane compound (68% corr) at135-145° C. The fractions are pooled to give 92 g monocyclopropanecompound of 100% purity.

Analytical data of E-Δ Farnesene:

1H-NMR (CDCl3, 400 MHz): 5.1 (2 m, 2 H), 4.6 (2 H), 2.2 (2 H), 2.1 (4H), 2.0 (2 H), 1.7 (s, 3 H), 1.6 (2 s, 6 H), 1.3 (1 H), 0.6 (2 H), 0.45(2 H) ppm. 13C-NMR (CDCl3, 400 MHz): 150.9 (s), 135.1 (s), 131.2 (s),124.4 (d), 124.1 (d), 106.0 (t), 39.7 (t), 35.9 (t), 26.7 (t), 25.7 (q),17.7 (q), 16.0 (d), 6.0 (t) ppm. GC/MS: 218 (2%, M+), 203 (5%, [M−15]+),175 (11%), 147 (31%), 134 (15%), 133 (20%), 121 (12%), 107 (55%), 95(16%), 93 (30%), 91 (20%), 82 (11%), 81 (33%), 79 (42%), 69 (100%), 67(22%), 55 (20%), 53 (21%), 41 (75%). IR (film): 3081 (w), 2967 (m), 2915(m), 2854 (m), 1642 (m), 1439 (m), 1377 (m), 1107 (w), 1047 (w), 1018(m), 875 (s), 819 (m), 629 (w). Anal. calcd. for C16H26: C, 88.00; H,12.00. Found: C, 87.80; H, 12.01.

C) Preparation of (7E)-4,8,12-trimethyltrideca-3,7,11-trien-1-ol((7E)-homofarnesol)

A mixture of (E)-(6,10-dimethylundeca-1,5,9-trien-2-yl)cyclopropane (E-ΔFarnesene) (1 g, 4.6 mmol), dodecane (0.2 g, 1.15 mmol, internalstandard) and L-(+)-tartaric acid (1 g, 6.9 mmol) in a pressure tube isheated under stirring at 150° C. After 18 h and complete conversion(according to GC) the mixture is poured on water (50 ml) and toluene (50ml). The phases are separated and the aqueous phase extracted withtoluene (50 ml). The combined organic layers are washed with conc.aqueous Na₂CO₃ (50 ml) and conc. NaCl (2×50 ml), dried over MgSO₄,filtered and evaporated under reduced pressure to give a brownish resin(1.35 g) which is mixed with 30% aqueous KOH (4.3 ml) and stirred at 25°C. for 2 h. GC analysis reveals formation of 96%(7E)-4,8,12-trimethyltrideca-3,7,11-trien-1-ol according to the internalstandard. E/Z ratio 68:22. The analytical data of the E-isomer areconsistent with the ones from the literature, see for example P.Kocienski, S. Wadman J. Org. Chem. 54, 1215 (1989).

Example 2

SHC Plasmid Preparation and Biocatalyst Production

SHC Plasmid Preparation

The gene encoding Alicyclobacillus acidocaldarius squalene hopenecyclase (AacSHC) (GenBank M73834, Swissprot P33247) was inserted intoplasmid pET-28a(+), where it is under the control of an IPTG inducibleT7-promotor for protein production in Escherichia coli. The plasmid wastransformed into E. coli strain BL21(DE3) using a standard heat-shocktransformation protocol.

Erlenmeyer Flask Cultures

For protein production were used either rich medium (LB medium) orminimal media. M9 is one example of minimal media, which weresuccessfully used.

Media Preparation

The minimal medium chosen as default was prepared as follows for 350 mlculture: to 35 ml citric acid/phosphate stock (133 g/l KH₂PO₄, 40 g/l(NH₄)₂HPO₄, 17 g/g citric acid.H₂O with pH adjusted to 6.3) was added307 ml H₂O, the pH adjusted to 6.8 with 32% NaOH as required. Afterautoclaving 0.850 ml 50% MgSO₄, 0.035 ml trace elements solution(composition in next section) solution, 0.035 ml Thiamin solution and 7ml 20% glucose were added.

SHC Biocatalyst Production (Biocatalyst Production)

Small scale biocatalyst production (wild-type SHC or SHC variants), 350ml culture (medium supplemented with 50 μg/ml kanamycin) were inoculatedfrom a pre-culture of the E. coli strain BL21(DE3) containing the SHCproduction plasmid. Cells were grown to an optical density ofapproximately 0.5 (OD_(650 nm)) at 37° C. with constant agitation (250rpm).

Protein production was then induced by the addition of IPTG to aconcentration of 300 μM followed by incubation for a further 5-6 hourswith constant shaking. The resulting biomass was finally collected bycentrifugation, washed with 50 mM Tris-HCl buffer pH 7.5. The cells werestored as pellets at 4° C. or −20° C. until further use. In general 2.5to 4 grams of cells (wet weight) were obtained from 1 litre of culture,independently of the medium used.

The fermentation was prepared and run in 750 ml InforsHT reactors. Tothe fermentation vessel was added 168 ml deionized water. The reactionvessel was equipped with all required probes (pO₂, pH, sampling,antifoam), C+N feed and sodium hydroxide bottles and autoclaved. Afterautoclaving, the following ingredients are added to the reactor:

-   -   20 ml 10× phosphate/citric acid buffer    -   14 ml 50% glucose    -   0.53 ml MgSO₄ solution    -   2 ml (NH₄)₂SO₄ solution    -   0.020 ml trace elements solution    -   0.400 ml thiamine solution    -   0.200 ml kanamycin stock

The reaction conditions are set as follows: pH=6.95, pO₂=40%, T=30° C.,Stirring at 300 rpm. Cascade: rpm setpoint at 300, min 300, max 1000,flow l/min set point 0.1, min 0, max 0.6. Antifoam control: 1:9.

The fermenter was inoculated from a seed culture to an OD_(650 nm) of0.4-0.5. This seed culture was grown in LB medium (+Kanamycin) at 37°C., 220 rpm for 8 h. The fermentation was run first in batch mode for11.5 h, where after was started the C+ N feed with a feed solution(sterilized glucose solution (143 ml H₂O+ 35 g glucose) to which hadbeen added after sterilization: 17.5 ml (NH₄)₂SO₄solution, 1.8 ml MgSO₄solution, 0.018 ml trace elements solution, 0.360 ml Thiamine solution,0.180 ml kanamycin stock. The feed was run at a constant flow rate ofapprox. 4.2 ml/h. Glucose and NH₄ ⁺ measurements were done externally toevaluate availability of the C- and N-sources in the culture. Usuallyglucose levels stay very low.

Cultures were grown for a total of approximately 25 hours, where theyreached typically and OD_(650 nm) of 40-45. SHC production was thenstarted by adding IPTG to a final concentration of approx. 1 mM in thefermenter (as IPTG pulse or over a period of 3-4 hours using an infusionsyringe), setting the temperature to 40° C. and pO₂ to 20%. Induction ofSHC production lasted for 16 h at 40° C. At the end of induction thecells were collected by centrifugation, washed with 0.1 M citricacid/sodium citrate buffer pH 5.4 and stored as pellets at 4° C. or −20°C. until further use.

Results Ia

In general, with all other conditions unchanged the specific activity ofthe produced biocatalyst was higher when a minimal medium was usedcompared with a rich medium.

Induction was carried out successfully at 30 or 37° C. It was noted thatwhen the induction was done at 40-43° C., a biocatalyst of higherspecific activity was obtained.

Results Ib

The following Table 1 shows for two examples the culture volume, opticaldensity and amount of cells both at induction start and induction end aswell as the amount of biomass collected (wet weight).

TABLE 1 cells cells Volume_(induction start) calculatedVolume_(induction end) collected (ml) OD_(650 nm induction start) (g)(ml) OD_(650 nm), _(induction end) (g) Example 1 273 40 10.9 342 55 28Example 2 272 44 12.0 341 57 23 OD_(650 nm) at inoculation: 0.45(Example 1) and 0.40 (Example 2). Starting volumes: 205 ml.

Wild type SHCamino acid sequence (GenBank M73834, Swissprot P33247)(SEQ ID No. 1)MAEQLVEAPAYARTLDRAVEYLLSCQKDEGYWWGPLLSNVTMEAEYVLLCHILDRVDRDRMEKIRRYLLHEQREDGTWALYPGGPPDLDTTIEAYVALKYIGMSRDEEPMQKALRFIQSQGGIESSRVFTRMWLALVGEYPWEKVPMVPPEIMFLGKRMPLNIYEFGSWARATVVALSIVMSRQPVFPLPERARVPELYETDVPPRRRGAKGGGGWIFDALDRALHGYQKLSVHPFRRAAEIRALDWLLERQAGDGSWGGIQPPWFYALTALKILDMTQHPAFIKGWEGLELYGVELDYGGWMFQASISPVWDTGLAVLALRAAGLPADHDRLVKAGEWLLDRQITVPGDWAVKRPNLKPGGFAFQFDNVYYPDVDDTAVVVWALNTLRLPDERRRRDAMTKGFRWIVGMQSSNGGWGAYDVDNTSDLPNHIPFCDFGEVTDPPSEDVTAHVLECFGSFGYDDAWKVIRRAVEYLKREQKPDGSWFGRWGVNYLYGTGAVVSALKAVGIDTREPYIQKALDWVEQHQNPDGGWGEDCRSYEDPAYAGKGASTPSQTAWALMALIAGGRAESEAARRGVQYLVETQRPDGGWDEPYYTGTGFPGDFYLGYTMYRHVFPTLALGRYKQAIERRVariant F601Y SHC amino acid sequence-variant with respect to SEQ ID No. 1(SEQ ID No. 2)MAEQLVEAPAYARTLDRAVEYLLSCQKDEGYWWGPLLSNVTMEAEYVLLCHILDRVDRDRMEKIRRYLLHEQREDGTWALYPGGPPDLDTTIEAYVALKYIGMSRDEEPMQKALRFIQSQGGIESSRVFTRMWLALVGEYPWEKVPMVPPEIMFLGKRMPLNIYEFGSWARATVVALSIVMSRQPVFPLPERARVPELYETDVPPRRRGAKGGGGWIFDALDRALHGYQKLSVHPFRRAAEIRALDWLLERQAGDGSWGGIQPPWFYALTALKILDMTQHPAFIKGWEGLELYGVELDYGGWMFQASISPVWDTGLAVLALRAAGLPADHDRLVKAGEWLLDRQITVPGDWAVKRPNLKPGGFAFQFDNVYYPDVDDTAVVVWALNTLRLPDERRRRDAMTKGFRWIVGMQSSNGGWGAYDVDNTSDLPNHIPFCDFGEVTDPPSEDVTAHVLECFGSFGYDDAWKVIRRAVEYLKREQKPDGSWFGRWGVNYLYGTGAVVSALKAVGIDTREPYIQKALDWVEQHQNPDGGWGEDCRSYEDPAYAGKGASTPSQTAWALMALIAGGRAESEAARRGVQYLVETQRPDGGWDEPYYTGTGYPGDFYLGYTMYRHVFPTLALGRYKQAIERRVariant F605W SHC nucleotide sequence  (SEQ ID No. 3)ATGGCTGAGCAGTTGGTGGAAGCGCCGGCCTACGCGCGGACGCTGGATCGCGCGGTGGAGTATCTCCTCTCCTGCCAAAAGGACGAAGGCTACTGGTGGGGGCCGCTTOTGAGCAACGTCACGATGGAAGCGGAGTACGTCCTCTTGTGCCACATTCTCGATCGCGTCGATCGGGATCGCATGGAGAAGATCCGGCGGTACCTGTTGCACGAGCAGCGCGAGGACGGCACGTGGGCCCTGTACCCGGGTGGGCCGCCGGACCTCGACACGACCATCGAGGCGTACGTCGCGCTCAAGTATATCGGCATGTCGCGCGACGAGGAGCCGATGCAGAAGGCGCTCCGGTTCATTCAGAGCCAGGGCGGGATCGAGTCGTCGCGCGTGTTCACGCGGATGTGGCTGGCGCTGGTGGGAGAATATCCGTGGGAGAAGGTGCCCATGGTCCCGCCGGAGATCATGTTCCTCGGCAAGCGCATGCCGCTCAACATCTACGAGTTTGGCTCGTGGGCTCGGGCGACCGTCGTGGCGCTCTCGATTGTGATGAGCCGCCAGCCGGTGTTCCCGCTGCCCGAGCGGGCGCGCGTGCCCGAGCTGTACGAGACCGACGTGCCTCCGCGCCGGCGCGGTGCCAAGGGAGGGGGTGGGTGGATCTTCGACGCGCTCGACCGGGCGCTGCACGGGTATCAGAAGCTGTCGGTGCACCCGTTCCGCCGCGCGGCCGAGATCCGCGCCTTGGACTGGTTGCTCGAGCGCCAGGCCGGAGACGGCAGCTGGGGCGGGATTCAGCCGCCTTGGTTTTACGCGCTCATCGCGCTCAAGATTCTCGACATGACGCAGCATCCGGCGTTCATCAAGGGCTGGGAAGGTCTAGAGCTGTACGGCGTGGAGCTGGATTACGGAGGATGGATGTTTCAGGCTTCCATCTCGCCGGTGTGGGACACGGGCCTCGCCGTGCTCGCGCTGCGCGCTGCGGGGCTTCCGGCCGATCACGACCGCTTGGTCAAGGCGGGCGAGTGGCTGTTGGACCGGCAGATCACGGTTCCGGGCGACTGGGCGGTGAAGCGCCCGAACCTCAAGCCGGGCGGGTTCGCGTTCCAGTTCGACAACGTGTACTACCCGGACGTGGACGACACGGCCGTCGTGGTGTGGGCGCTCAACACCCTGCGCTTGCCGGACGAGCGCCGCAGGCGGGACGCCATGACGAAGGGATTCCGCTGGATTGTCGGCATGCAGAGCTCGAACGGCGGTTGGGGCGCCTACGACGTCGACAACACGAGCGATCTCCCGAACCACATCCCGTTCTGCGACTTCGGCGAAGTGACCGATCCGCCGTCAGAGGACGTCACCGCCCACGTGCTCGAGTGTTTCGGCAGCTTCGGGTACGATGACGCCTGGAAGGTCATCCGGCGCGCGGTGGAATATCTCAAGCGGGAGCAGAAGCCGGACGGCAGCTGGTTCGGTCGTTGGGGCGTCAATTACCTCTACGGCACGGGCGCGGTGGTGTCGGCGCTGAAGGCGGTCGGGATCGACACGCGCGAGCCGTACATTCAAAAGGCGCTCGACTGGGTCGAGCAGCATCAGAACCCGGACGGCGGCTGGGGCGAGGACTGCCGCTCGTACGAGGATCCGGCGTACGCGGGTAAGGGCGCGAGCACCCCGTCGCAGACGGCCTGGGCGCTGATGGCGCTCATCGCGGGCGGCAGGGCGGAGTCCGAGGCCGCGCGCCGCGGCGTGCAATACCTCGTGGAGACGCAGCGCCCGGACGGCGGCTGGGATGAGCCGTACTACACCGGCACGGGCTTCCCAGGGGATTGGTACCTCGGCTACACCATGTACCGCCACGTGTTTCCGACGCTCGCGCTCGGCCGCTACAAGCAAGCCATCGAGCGCAGGTGAVariant F605W SHC amino acid sequence-variant with respect to SEQ ID No. 1(SEQ ID No. 4)MAEQLVEAPAYARTLDRAVEYLLSCQKDEGYWWGPLLSNVTMEAEYVLLCHILDRVDRDRMEKIRRYLLHEQREDGTWALYPGGPPDLDTTIEAYVALKYIGMSRDEEPMQKALRFIQSQGGIESSRVFTRMWLALVGEYPWEKVPMVPPEIMFLGKRMPLNIYEFGSWARATVVALSIVMSRQPVFPLPERARVPELYETDVPPRRRGAKGGGGWIFDALDRALHGYQKLSVHPFRRAAEIRALDWLLERQAGDGSWGGIQPPWFYALTALKILDMTQHPAFIKGWEGLELYGVELDYGGWMFQASISPVWDTGLAVLALRAAGLPADHDRLVKAGEWLLDRQITVPGDWAVKRPNLKPGGFAFQFDNVYYPDVDDTAVVVWALNTLRLPDERRRRDAMTKGFRWIVGMQSSNGGWGAYDVDNTSDLPNHIPFCDFGEVTDPPSEDVTAHVLECFGSFGYDDAWKVIRRAVEYLKREQKPDGSWFGRWGVNYLYGTGAVVSALKAVGIDTREPYIQKALDWVEQHQNPDGGWGEDCRSYEDPAYAGKGASTPSQTAWALMALIAGGRAESEAARRGVQYLVETQRPDGGWDEPYYTGTGFPGDWYLGYTMYRHVFPTLALGRYKQAIERR

Example 3

Bioconversion of 7E, 3E/Z-Homofarnesol Mixture

Bioconversion was undertaken using the following reaction conditions:

The reaction (150.1 g total volume) run in 0.1 M citric acid/sodiumcitrate buffer pH 5.4 in an InforsHT 750 ml fermenter contained 146 g/ltotal homofarnesol using a homofarnesol substrate, which was a mixtureof 7E,3E:7E,3Z of 86:14, 250 g/l cells (formed in accordance with themethod of Example 2, fermentation) and 1.55% SDS. The reaction was runat 35° C. with constant stirring (900 rpm), pH control was done using 10to 40% citric acid in water.

The reaction mixture was subjected to isolation and purification stepsas set forth in Example 4, below.

Example 4

Downstream Processing Procedure

A reaction mixture formed from the bioconversion of 7E,3E/Z-homofarnesol (86:14 3E:3Z) was subjected to steam distillation. Thedistillate was collected as a biphasic mixture. The organic phase wasretained and the aqueous phase discarded. The composition of the organicphase was analysed by GC and the results shown in the Table 2 below (see“crude”).

The organic phase was then concentrated to dryness. Ethanol was thenadded to the crude, dried product and the mixture warmed until theproduct was dissolved. At room temperature water is slowly added and(−)-Ambrox crystallizes under occasional stirring and cooling in an icebath.

Table 1 shows the GC analytics results for the crystallized product. Thedata show a strong enrichment of (−)-Ambrox, with practically noby-products (a), (b) or (c) being found in the crystallized sample.

It should be noted that in Table 2, “a”, “b” and “c” refer to compound(II), compound (IV) and compound (III) respectively. “EZH” and “EEH”refer to 7E,3Z-homofarnesol and 7E,3E-homofarnesol respectively.

TABLE 1 Peak area (GC) (—)-Ambrox a b c (—)-Ambrox EZH EEH (%) Crude215073 190376 588769 6751605 13429 14184 86.9 Crystallized 10088 895164625 9032941 0 0 99.1

Example 5

Extraction of the solid phase of the reaction broth:

Given that (−)-Ambrox is not soluble in water and is not liquid attemperatures below approx. 75° C., these properties were taken aspossible advantages to extract the product from the solid phase of thebiotransformation using either water-miscible solvents (e.g. ethanol)and water-immiscible solvents (e.g. toluene).

200 ml reaction broth was centrifuged to separate the solid from theliquid (aqueous) phase (Sorvall GS3, 5000 rpm, 10 min, 10° C.). Thisseparated approx. 80 ml solid pellet from approx. an 120 ml liquidphase. Analysis (Gas chromatography) of the aqueous phase after MTBEextraction showed that it contained not more than approx. 0.3% of the(−)-Ambrox initially present in the 200 ml reaction broth. Toluene andethanol 99% were used for extracting (−)-Ambrox from the solid phase.

Toluene Extraction:

80 ml solid phase was extracted 6× with 45 ml toluene (approx. ½ solidphase volume, vigorous shaking for 30 s, centrifugation (Sorvall GS3,5000 rpm, 10 min, 10° C.). The solvent phase was analyzed with GC forits (−)-Ambrox content. Over 99.5% of (−)-Ambrox initially present inthe reaction broth was extracted with 6 extractions representing a totaltoluene vol. of 1.35× the initial whole reaction broth volume (200 ml)or 3.4× the vol. of the solid phase.

Ethanol Extraction:

80 ml solid phase was extracted (Infors Multifors HT, 35° C., 1000 rpm,30 min) with approx. 160 ml (2 vol.) ethanol 99%, followed bycentrifugation. (−)-Ambrox did not crystallize during the extractionprocedure. After 4 washes (total 640 ml ethanol, i.e. 3.2× the initialwhole reaction broth volume or 8× the volume of the solid phase), about99% of (−)-Ambrox initially present in the reaction broth was recovered.Sufficient ethanol is required in the first extraction step to prevent(−)-Ambrox crystallization (solubility in ethanol). When only 1 or ½ volof the solid phase was used in the first extraction step, a sticky pastewas obtained, which was difficult to handle and (−)-Ambrox crystallizedas needles on the pellet during centrifugation. Temperature appeared asnot being the factor responsible for this crystallization (extractionand centrifugation tested at room temperature and approx. 35° C.-40°C.).

The (−)-Ambrox concentration in the ethanol phase as well as theethanol/water ratio of the liquid phase (residual moisture of the solidphase) appeared to be responsible for crystal formation. It was howevernoted that it was possible to reduce the volume of ethanol to 1 vol ofthe solid phase.

As (−)-Ambrox is not in the liquid phase at room temperature, itseparates with the biomass and can be extracted with an organic solvent(e.g. a water-miscible solvent (e.g. ethanol) or a water-immisciblesolvent (e.g. toluene). The centrifugation step that separates the(−)-Ambrox into the solid phase of the reaction mixture is advantageousbecause it reduces the amount of solvent required to extract (−)-Ambrox.

Example 6

Sensory Analysis

Purpose: to carry out a sensory analysis of (−)-Ambrox and the compounds(II), (III) and (IV) formed in the crude material and in thecrystallised material.

Biotransformation of E,E-homofarnesol results in (−)-Ambrox, andcompound (IV).

Biotransformation of E,Z-homofarnesol results in the macrocyclic ethercompound (II) and epi-Ambrox compound (III).

A crude mixture of (−)-Ambrox comprises the desired (−)-Ambrox, compound(II), (III) and (IV) present in an amount of 87.1 wt %, 2.8 wt %, 2.5 wt% and 7.6 wt % respectively.

When a crude mixture is selectively crystallised (lab scale), thecrystallised material has the same components as the crude mixture, butthey are present in an amount of 99.1 wt %, 0.1 wt %, 0.1 wt % and 0.7wt % respectively.

The Sensory Analytical Results were as follows:

(−)-Ambrox: Odour Threshold 0.2 ng/l.

Compound (IV): weak, IsoE, woody, GC-detection threshold 5-10 ng.

Compound (II): “odorless” (GC-threshold >500 ng).

Compound (III): GC-threshol about 10× higher than (−)-Ambrox (circa 2ng).

The sensory analysis of the 3 by-products (compounds II, III and IV)indicates a weaker odour than that from (−)-Ambrox. In fact, theepi-ambrox (Compound III) odor is about 10 fold weaker than (−)-Ambroxsuggesting that it is essentially odorless.

Example 7

Ambrox Recovery by Steam Extraction

Resulting Purity of the Crude (Steam Extracted) and Crystallized(−)-Ambrox

The biotransformation of EE:EZ-homofarnesol 86:14 provided a reactionmixture that was steam extracted. The steam distillate was collected asa biphasic mixture. The organic phase was retained and the aqueous phasediscarded. The composition of the organic phase was analysed by GC andthe results shown in the Table below (see “crude”). The organic phasewas then concentrated to dryness. Ethanol was then added to the crude,dried product and the mixture warmed until the product was dissolved. Atroom temperature water is slowly added and (−)-Ambrox crystallizes underoccasional stirring and cooling in an ice bath.

The tabulated data also shows the GC analytics results for productsobtained after the steam extraction/distillation step (“crude”) and thecrystallized product ((−)-Ambrox). The references in the Table to “EZH”and “EEH” refer to (3Z,7E)-homofarnesol and 7E,3E-homofarnesolrespectively.

The tabulated data below indicates that the particular starting material(EEH:EZH 86:14) produces the desired end product (−)-Ambrox and a veryspecific mixture of by-products (II, IV and III) using the WT SHC enzymeor a SHC derivative. The data for the selective crystallization show astrong enrichment of (−) Ambrox, with practically no by-products (II),(IV) or (III) being found in the crystallized sample. Accordingly, thisEE:EZ mixture provides an olfactively pure (−)-Ambrox product, which isselectively crystallised in a relatively straightforward andcost-effective matter.

TABLE shows the GC analytics results for the crystallized product. Peakarea (GC) Ambrox (II) (IV) (III) Ambrox EZH EEH (%) Crude 215073 190376588769 6751605 13429 14184 86.9 Crystallized 10088 8951 64625 9032941 00 99.1

Steam extraction/filtration are environmentally friendly methods forisolating (−)-Ambrox because it offers a convenient solvent-freeisolation of (−)-Ambrox with an associated inactivation of thebiocatalyst.

The (−)-Ambrox produced using the bioconversion reaction may beextracted using solvent from the whole reaction mixture (e.g. using awater-immiscible solvent or by steam extraction/distillation or byfiltration) or from the solid phase (e.g. using a water misciblesolvent) using methods which are known to those skilled in the art.

1. A method of isolating and purifying (−)-Ambrox from a reactionmixture, comprising one or more of the compounds (II), (III) and (IV)


2. The method according to claim 1 comprising the step of selectivelycrystallizing (−)-Ambrox from a mixture comprising one or more of thecompounds (II), (III) or (IV).
 3. A method of improving or enhancing theodour of (−)-Ambrox comprising the step of separating (−)-Ambrox from amixture comprising one or more of the compounds (II), (III) or (IV)

by selective crystallization of (−)-Ambrox from the mixture, such thatafter the step of separating, (−)-Ambrox contains none, or onlyolfactory acceptable amounts, of the compounds (II), (III) or (IV). 4.The method according to claim 1, wherein the reaction mixture is free,or is substantially free, of homofarnesol.
 5. The method according toclaim 2, wherein the crystallizing solvent is selected from the groupconsisting of water, methanol, acetone, petroleum ether, hexane, t-butylmethyl ether, THF and ethyl acetate ethanol, toluene and mixturesthereof.
 6. The method according to claim 5, wherein the crystallizingsolvent is an ethanol water mixture.
 7. The method according to claim 1,wherein the reaction mixture is formed as a result of anenzyme-catalyzed cyclization of homofarnesol comprising a mixture of7E,3E and 7E,3Z homofarnesol geometric isomers of homofarnesol, whereinthe reaction is carried out in the presence of a recombinantmicroorganism expressing the gene encoding the enzyme.
 8. The methodaccording to claim 7, wherein the reaction mixture of 7E,3E and 7E,3Zhomofarnesol is enriched in the 7E,3E geometric isomer.
 9. The methodaccording to claim 7, wherein the reaction mixture of 7E,3E and 7E,3Zhomofarnesol consists of 7E,3E and 7E,3Z homofarnesol and no othergeometric isomers of homofarnesol.
 10. The method according to claim 7,wherein the weight ratio of the 7E,3E isomer to 7E,3Z isomer is at least80:20.
 11. The method according to claim 7, wherein the enzyme is awild-type squalene hopene cyclase or a variant of the wild-type squalenehopene cyclase.
 12. A perfume ingredient consisting of (−)-Ambrox andolfactory acceptable amounts of one or more of the compounds (II), (III)or (IV)


13. The perfume ingredient according to claim 12, comprising crystalline(−)-Ambrox
 14. A perfume composition comprising (−)-Ambrox and at leastone other perfume ingredient, wherein said perfume composition containsolfactory acceptable amounts of one or more of the compounds (II), (III)or (IV)


15. The method according to claim 3, wherein the reaction mixture isfree, or is substantially free, of homofarnesol.
 16. The methodaccording to claim 3, wherein the crystallizing solvent is selected fromthe group consisting of water, methanol, acetone, petroleum ether,hexane, t-butyl methyl ether, THF and ethyl acetate ethanol, toluene andmixtures thereof.
 17. The method according to claim 16, wherein thecrystallizing solvent is an ethanol water mixture.
 18. The methodaccording to claim 3, wherein the mixture is formed as a result of anenzyme-catalyzed cyclization of homofarnesol comprising a mixture of7E,3E and 7E,3Z homofarnesol geometric isomers of homofarnesol, whereinthe reaction is carried out in the presence of a recombinantmicroorganism expressing the gene encoding the enzyme.
 19. The methodaccording to claim 18, wherein the reaction mixture of 7E,3E and 7E,3Zhomofarnesol is enriched in the 7E,3E geometric isomer.
 20. The methodaccording to claim 18, wherein the reaction mixture of 7E,3E and 7E,3Zhomofarnesol consists of 7E,3E and 7E,3Z homofarnesol and no othergeometric isomers of homofarnesol.
 21. The method according to claim 18,wherein the weight ratio of the 7E,3E isomer to 7E,3Z isomer is at least80:20.
 22. The method according to claim 18, wherein the enzyme is awild-type squalene hopene cyclase or a variant of the wild-type squalenehopene cyclase.
 23. The method according to claim 10, wherein the weightratio of the 7E,3E isomer to 7E,3Z isomer is at least 90:10.
 24. Themethod according to claim 10, wherein the weight ratio of the 7E,3Eisomer to 7E,3Z isomer is at least 95:5.